The propose addresses a central problem in developmental biology: how cells in different regions of the embryo are assigned to different developmental pathways. In the Drosophila embryo, developmental pathways are specified combinatorially by the localized expression of segmentation genes. Several in vivo approaches are proposed to investigate how segmentation gene expression is localized, using as a model system the spatial regulation of the engrailed gene by pair-rule genes. (i) Epistasis studies will be used to determine the regulatory pathways of engrailed. The RNA and protein used to determine the regulatory pathways of engrailed. The RNA and protein expression of engrailed will be measured in double mutants of its regulators, using in situ hybridization and immunolabeling. (ii) Expression domains of engrailed and its regulatory interactions. (iii) The effects of ectopic expression of pair-rule genes on the expression of engrailed will be measured to determine if the observed changes are consistent with a combinatorial model of regulation. The genes will be placed under the control of a heat-shock promoter and expressed in embryos using germ-line transformation. (iv) A functional dissection of the pair-rule regulators of engrailed will be performed using in vivo assay in the embryo. Chimeras of different pair-rule genes will be tested for their ability to regulate engrailed expression when they are placed under the control of either a heat-shock promoter, or the promoter of one of the contributing parental genes. By examining whether the chimera behaves like one of the parental genes, it should be possible to determine which parts of the pair-rule gene are required for the specific regulation of engrailed. These experiments will provide an in vivo test of the functional importance of evolutionarily- conserved motifs such as the homeobox and paired box. (v) An inhibitor assay, in which short-lived regulators of segmentation genes are depleted by blocking general protein synthesis, will be used to describe the spatial regulation of several pair-rule and segment-polarity genes.